Regulation of expression of the adenovirus 2 genome will be studied using combined biochemical and genetic methods. The effects of inhibitors of protein synthesis and mutant early viral polypeptides on the biosynthesis of mRNA will be investigated. Polyadenylated and non-polyadenylated nuclear RNA species will be identified and localized within the genome by hybridization to restriction enzyme fragments of viral DNA. Pulse label and chase experiments will be performed to study the relationship of RNA precursors, processing intermediates, and cytoplasmic message. A system using isolated nuclei which faithfully mimics in vivo conditions will be developed to study RNA transcription and processing. Conditional-lethal temperature-sensitive mutants of adenovirus 2 will be constructed by in vitro mutagenesis of specific viral DNA restriction enzyme fragments, and in vivo recombination of mutagen-treated fragments and wild type DNA to generate mutant genomes. Temperature-sensitive mutants defective in early viral functions will be isolated and characterized with regard to cell transformation and expression of early viral genes.